Recurrent spontaneous Escherichia coli meningitis in an adult: a case report

Abstract Objectives The aim of this study was to characterize an unusual case of spontaneous, community-acquired Escherichia coli meningitis in an adult presenting to a general hospital in Kenya, where initial clinical recovery was followed by reinfection with an MDR, hospital-acquired strain. Patient and methods An adult presented to a hospital in Kenya with meningitis symptoms. E. coli was cultured from CSF. Treatment with ceftriaxone was successful; however, the patient relapsed a few days later. E. coli was cultured from CSF and blood during the reinfection episode, though the patient died during admission. We sequenced the isolates using Illumina MiSeq and performed antimicrobial susceptibility testing, fitness and virulence assays on the bacteria. Results The E. coli isolates from the two episodes were found to be distinct: the initial strain was ST88, serotype O8 H17 while the subsequent episode was caused by an ST167, serotype O101 H5 MDR strain. The ST88 strain was susceptible to all drugs except ampicillin and amoxicillin/clavulanate while the ST167 strain was MDR, including to all β-lactam drugs due to the presence of the carbapenemase gene blaNDM-5. The hospital-acquired ST167 strain was also resistant to newer drugs such as cefiderocol and eravacycline, which are currently not available locally, and had overall lower fitness and virulence in vitro compared with the initial infecting strain. Conclusions Though less fit and virulent in vitro, the MDR strain was fatal, suggesting that host factors, rather than bacterial virulence, may have been of greater importance in this patient’s outcome.


Introduction
The spread of MDR infections is a worldwide health concern and its impact on mortality is particularly high in resource-limited settings 1 where several factors, including lack of access to reliable laboratory infrastructure for microbiological analysis, delay access to optimally targeted medication.
Escherichia coli causes a variety of diseases and is a common cause of urinary tract infections, meningitis, sepsis and abdominal infections, and resistance in the pathogen is a leading cause of resistance-associated mortality. 1 In adults, spontaneous community-acquired E. coli meningitis is rare and carries a high mortality. It is most often associated with neurosurgical procedures or head trauma, 2,3 with other cases reported as secondary dissemination from a urinary tract or gastrointestinal tract infection. 4,5 Several risk factors have been identified, including advanced age, chronic alcoholism, liver cirrhosis, immunocompromised conditions due to HIV infection or immunosuppressive therapy and diabetes mellitus or, in some cases, strongyloidiasis infection. 4,[6][7][8] In this paper, we describe a case of spontaneous onset of meningeal infection by a sensitive E. coli strain followed by reinfection with an MDR strain. Both isolates were characterized using WGS and in vitro fitness assays to evaluate the contribution of bacterial versus host factors.
Separate approval was acquired for further data analysis described (SERU 3748). Written informed consent to participate in the surveillance study was obtained from the case on admission to hospital. Publication consent for the case report was obtained from the next of kin.

Case report
A middle-aged male patient presented to a county general hospital in rural coastal Kenya with a 1 week history of fever, diarrhoea, vomiting, headache and confusion. He reported progressive weight loss over the preceding months with loss of appetite and poor nutrition. He had a history of excessive alcohol use and cigarette smoking but no other significant past medical history. He denied attending any other healthcare facilities in the preceding 6 months and tested negative for HIV. At admission, blood cultures were taken, and a lumbar puncture was performed: the CSF was noted to be turbid, with an elevated protein concentration (3.55 g/L), and WBC count (>999 cells/µL), and a glucose level below the detection limit of the assay. Direct staining of the CSF showed Gram-negative rods, which were confirmed to be E. coli, susceptible by disc diffusion to gentamicin, amikacin, chloramphenicol, trimethoprim/ sulfamethoxazole, ceftriaxone, ciprofloxacin, ceftazidime, cefoxitin, cefotaxime and imipenem, and with reduced susceptibility to ampicillin and amoxicillin/clavulanate. The blood culture was negative after 72 h. The patient was admitted to a general ward and was treated with 2 g IV ceftriaxone twice daily for 3 weeks. He also received diazepam and IV thiamine to prevent delirium tremens and other overt signs of alcohol withdrawal. He made a full clinical recovery with resolution of fever and meningism and was discharged from the general service. However, he delayed leaving the hospital and was retained in the general ward due to financial reasons. Three days after completing the ceftriaxone course, he once more became unwell with fever and reduced level of consciousness. He had a repeat lumbar puncture and blood culture, and ceftriaxone was restarted empirically. This time, the CSF was clear on examination, with a protein concentration of 2.67 g/L, WBC count of 760 cells/µL and glucose again below the detection limit of the assay. Within 72 h, both the blood culture and CSF grew a carbapenem-resistant E. coli susceptible to gentamicin, amikacin, co-trimoxazole and chloramphenicol. He was switched to chloramphenicol but unfortunately died 2 days later.
Three E. coli strains, from CSF culture at admission (ECO-1) and during recurrence (ECO-2) and from blood during recurrence (ECO-3), were obtained from the patient and studies were undertaken to characterize virulence and resistance factors.

Growth curve assay
The assay was adapted from previously published methods. 20,21 Overnight cultures of the strains were prepared in normal saline to a 0.5 McFarland suspension and diluted 1:1000 in either fresh standard LB media (standard condition) or 5% w/v sodium chloride-supplemented LB media (stressed condition). The mixture was incubated at 37°C in triplicate in a microplate reader (Synergy H1, BioTek, USA) and growth was measured every 30 min for 15 h at an OD of 600 nm (OD 600 ), with agitation for 5 min prior to each measurement. The assay was performed on three separate days and the averages used to generate growth curves.

In vitro mixed growth competition assay
The assay measured the in vitro competition between carbapenemresistant and -susceptible strains from CSF and was adapted from previously published protocols. 20,22,23 The strains were each grown to exponential phase (4 h) in standard LB or 5% w/v salt-supplemented LB media and incubated at 35°C ± 2°C. The exponentially growing cells were diluted to 0.5 McFarland suspension and 10 µL each of resistant and susceptible strain was added to 10 mL of fresh medium (standard or 5% salt media). The mixture was incubated at 37°C while shaking at 180 rpm for 24 h. Serial 10-fold dilutions of the mixture were plated on Mueller-Hinton agar (MHA) plates without and with 0.25 mg/L meropenem and incubated overnight at 35°C ± 2°C. The cfu/mL for the resistant strain, ECO-2, was calculated from the meropenem-containing plate, and the cfu/mL for the susceptible strain, ECO-1, was determined by subtracting the cfu from plates with antibiotic from those without. The competition index (CI) was defined as the ratio between cfu/mL values of carbapenem-resistant and -susceptible strains. The CI values were calculated for each independent competition assay performed on three separate days and the mean CI determined.

Serum bactericidal assay
Normal human serum from healthy volunteers was pooled and frozen at −70°C until use. The assay was adapted from published methods. 21,24,25 Strains were grown overnight in standard LB broth, then standardized to 0.5 McFarland and grown at 35°C ± 2°C for 4 h to achieve the exponential growth state. One millilitre of the growing cells was aliquoted and spun at 10 000 rpm for 10 min to pellet the bacteria. The pellet was resuspended and washed twice with PBS. The cells were reconstituted in 1 mL of PBS, and 100 µL was added to 900 µL of 40% serum in sterile PBS. The mixture was incubated at 37°C for 3 h with shaking at 180 rpm. Immediately and at 45 min intervals over the 3 h period, an aliquot was serially diluted 10-fold, plated on MHA and incubated overnight at 35°C ± 2°C, and viable Amulele et al.
cfu/mL value was determined. The assay was performed on three separate days and the mean used to determine serum sensitivity of the strains.

Hospital-acquired strains were highly resistant, including to newer antimicrobials
At admission, the first strain identified as a community-acquired E. coli strain, ECO-1, was susceptible to all drugs tested except ampicillin and amoxicillin/clavulanate, to which it had reduced susceptibility (intermediate) ( Table 1). The reinfection episode was due to hospital-acquired MDR isolates, (ECO-2 from CSF and ECO-3 from blood), which were susceptible to gentamicin, amikacin, chloramphenicol and colistin. The MDR strains were found to be resistant to eravacycline, cefiderocol, imipenem/relebactam and meropenem/vaborbactam, despite no known exposure to any of these compounds.
The hospital-acquired strains possessed several resistance genes including β-lactamases bla NDM-5 , bla CTX-M-15 and bla OXA-1 , aminoglycoside resistance genes aadA2, aac(6′)-Ib-cr and aac(3)-lla and genes conferring resistance to other antimicrobials and cleaning compounds catB3, dfrA12, sul1, tet(A), mph(A), mdf(A) and qacE (Table 1). In addition, point mutations in gyrase (gyrA) and topoisomerase genes (parC and parE) conferring resistance to fluoroquinolones were present. The ECO-1 strain had two resistance-associated genes, sul2 and tet(B), which confer resistance to sulfamethoxazole and tetracycline, respectively. The strain was susceptible to sulfamethoxazole/trimethoprim in vitro despite the presence of sul2. Phenotypic susceptibility to tetracycline was not tested.

Community-acquired strain was unrelated to hospital-acquired strains
Genomic analysis revealed the community-acquired strain (ECO-1) to be ST88, serotype O8:H17, while the hospital-acquired CSF (ECO-2) and bacteraemia strains (ECO-3) were both ST167, serotype O101:H5. There were also differences in the plasmids found in strains with one plasmid, Col8282, detected in the community-acquired strain and four plasmids, Col(BS512), ColpVC, IncFIA and IncFII, detected in the hospital-acquired strains.

Hospital-acquired strain was less virulent and less fit than community-acquired strain
The community-acquired strain had additional virulence genes compared with the hospital-acquired strain, with the presence of genes involved in iron acquisition (iucC, iutA, fyuA, ireA, iroN, sitA, irp2), serum resistance (iss, traT), toxicity (hlyF), adhesion (papC, papA_F20, lpfA), outer membrane protease (ompT), bacteriocin (cvaC) and miscellaneous genes (hra, tsh, etsC and mchF). The resistant strains possessed far fewer virulence genes, with only iss and traT (involved in serum resistance) and hra, a heat-resistant agglutinin, identified in the genome. These three genes were also present in the community-acquired strain. None of the strains possessed the Shiga toxin gene.
In individual growth curves in standard LB broth, the susceptible strain, ECO-1, grew slightly faster than ECO-2 and ECO-3 (resistant strains), and the difference was amplified in 5% salt media, with detectable bacterial amplification at 5.5 h for ECO-1 versus 8.5 h for ECO-2 and ECO-3 ( Figure 1). Pairwise competition between the CSF strains demonstrated that under standard conditions, ECO-2 was less fit, with a mean CI of 0.695 (SD = 0.25); however, in 5% salt it was the ECO-1 strain that was less fit (mean CI 2.33, SD = 1.44). ECO-2 and ECO-3 showed reduced survival in normal human serum compared with ECO-1, with a reduction in viable bacterial count from 8 log 10 cfu/mL to less than 6 log 10 cfu/mL by 45 min and through the assay, compared with ECO-1, which was unaffected at 45 min and increased to 10 log 10 cfu/mL by 180 min (Figure 2).

Discussion
Here we present an interesting case of recurrent meningitis in an adult with two distinct E. coli strains, one of which was carbapenem resistant. To the best of our knowledge, this is the first nonsurgical case where reinfection has been reported with an entirely different strain of E. coli. Spontaneous community-acquired E. coli meningitis in immunocompetent adults is rare, attributed to predisposing risk factors and/or occurring secondary to a distant infection. Though published data on adult meningitis in Africa are limited, non-meningococcus and non-pneumococcus bacteria including E. coli accounted for 10% of all cases in adolescents and adults reported from 1990 to 2013 in Malawi. 26 The community-and hospital-acquired strains had different antibiograms, suggesting that they were unrelated. This was confirmed by WGS, with the initial community-acquired strain being ST88 and the hospital-acquired isolate belonging to an unrelated ST, ST167. They also had different resistance and virulence gene profiles and possessed different plasmid types. This was, therefore, a new infection rather than in vivo acquisition of resistance in the initial strain.
Previous studies have identified chronic alcoholism as a risk factor for Gram-negative meningitis. 4 This may explain the initial presentation in this patient, who had a history of excess alcohol intake, although he had not progressed to liver impairment. While no other risk factors were identified, he had complained of symptoms suggestive of gastroenteritis prior to his admission. It is therefore possible that there was an intrabdominal source that was not identified during his stay, as no gastrointestinal investigations were carried out because his symptoms were ascribed to meningitis and sepsis.
Alternatively, the patient may have had a mixed infection consisting of both carbapenem-resistant and -susceptible strains, acquired prior to admission to hospital. If the resistant strain was present in low density it may have been missed during selection of a single colony for susceptibility testing. Treatment with a broad-spectrum cephalosporin may have resulted in initial control of the infection, reduction in bacterial load and subsequent improvement in the patient's condition, while concurrently providing the selective pressure that allowed the resistant strain to survive. However, given the patient's lack of prior hospital exposure, this is less likely given the relative rarity of carbapenem resistance in the community in Kenya, and the MDR strain was therefore more likely to be hospital acquired.
The community-acquired strain possessed multiple virulence genes, which may explain the initial infection. A recent study in Recurrent E. coli meningitis in an adult Table 1 Amulele et al.
France showed that patients who died of E. coli meningitis were more likely to have been infected with a more virulent strain compared with survivors. 8 Moussiegt et al. 8 also reported that acquired resistance genes in the meningitis strains was rare, a phenomenon observed in the initial infecting strain in our case study. The hospital-acquired strain possessed very few virulence genes, and the lack of virulence was confirmed in vitro, with reduced susceptibility to serum from healthy humans. In competition assays, it was less fit than the community-acquired strain, except in pairwise competition in 5% salt, where there was no significant difference in growth between the strains, suggesting similar responses to this specific stressor. Despite this, it was able to infect the patient with a lethal outcome, suggesting that in this case, host factors played a significant role in the pathogenesis of infection in this patient. We postulate, therefore, that debilitation due to chronic alcohol intake and poor nutrition, together with a possible intra-abdominal source of infection, caused initial infection in this patient, who unfortunately become colonized with a carbapenem-resistant strain during his hospital stay. Those same risk factors, together with the selective pressure from the cephalosporin administered for his first infection, subsequently predisposed him to a second infection, this time with the resistant strain. Additionally, although the patient did not have HIV, underlying immune dysfunction due to poor nutrition 27,28 and chronic alcohol use 29 cannot be ruled out, with the carbapenemresistant organism behaving as an opportunistic pathogen.
Discordance between genotypic and phenotypic results was observed for the aminoglycosides, chloramphenicol and sulfamethoxazole/trimethoprim. The catB3 gene in the resistant strain, which confers chloramphenicol resistance, had only 70% coverage to the reference gene used in ResFinder and thus may not have yielded a fully functioning protein; however, in the case of aminoglycosides the genes detected may confer resistance to other aminoglycosides such as spectinomycin while the presence of aac(6′)-Ib-cr does not necessarily confer resistance to amikacin, gentamicin or ciprofloxacin. 30 The reasons for the susceptibility to sulfamethoxazole/trimethoprim despite the presence of the sul2 gene in the susceptible E. coli strain remain unknown, though likely causes may include changes in the promoter region affecting expression or the less-known antibiotic resistance gene-silencing concept previously reported in animal studies. 31 In some strains, drug susceptibility was observed despite the plasmid possessing intact ORFs and promoter regions for bla OXA-2 , aadA1, sul1 and tet(A) genes similar to WTs. In such cases, the transcripts were found to be lacking, suggesting that resistance gene expression could be switched off, though the mechanisms are unknown. However, this phenomenon was potentially reversible when the plasmid was introduced into a new host bacterium, implying that chromosomal factors rather than plasmid ones were responsible.
The patient was treated with chloramphenicol but died 2 days later. While a partial chloramphenicol resistance gene was identified in the resistant strain, phenotypic susceptibility testing predicted in vivo efficacy, but by the time testing was complete he had been on ineffective therapy with ceftriaxone for 3 days. An alternative treatment regimen with a combination of chloramphenicol and amikacin may have been beneficial in this case; however, even with treatment that is concordant with in vitro susceptibility, more than a third of spontaneous E. coli meningitis patients die. 4,5 It is likely that the underlying factors contributing to initial infection also contributed to death in this case.
A final, important aspect of this case is the extensive resistance observed in the hospital-acquired strain. Carbapenems are generally unavailable in the public sector in Kenya, 32,33 although they are widely used in private hospitals, 34  Even more concerning is the resistance to eravacycline, cefiderocol and the newer β-lactam/inhibitor combinations. To the best of our knowledge, these drugs are not available in Kenya; Recurrent E. coli meningitis in an adult however, resistance to eravacycline and tigecycline (closely related to eravacycline) due to the presence of plasmid-encoded variants of the tet(X) gene: tet(X4) 35,36 and tet(X5), 37 as well as mutations in the efflux pump AdeABC, 38 have been reported in Gram-negative bacteria from Asia. Resistance to cefiderocol has been associated with mutations in siderophore receptor gene, cirA, in Enterobacterales, 39,40 with a recent study 41 reporting the concomitant presence of β-lactamases such as bla NDM and mutations in cirA resulting in resistance to the antibiotic. We did not detect the presence of tet(X) genes in the MDR strains but it is likely that mutations in the efflux pumps or the siderophore may be involved in resistance. The presence of resistance to drugs not currently in use in the country is deeply concerning and illustrates the ongoing challenges of antimicrobial resistance. Surveillance to detect resistance to new antibiotics is necessary and a One Health approach that incorporates the animal and agricultural sectors can help detect reservoirs for MDR bacteria with clinical implications.